This research proposal focuses on erythropoietin activated signaling pathways. Based on previous studies, we hypothesize that Epo activates multiple signaling pathways, and among them, P13 kinase signaling is involved in the initiation of c-myc transcription. We also hypothesize that the phospholipase C-y (PLCy)/phosphoinositide-dependent protein kinase 1 (PDK1) /PKC-e/unidentified kinase/ MAP kinase kinase (MEK)/MAP kinase (ERK) cascade is involved in the elongation of c-myc and is also involved in the regulation of c-fos by the Elk transcription factor. Also, the Ras-Raf-I pathway regulates c-fos through serum response factor (SRF) or AP-1 transcription factor. The effectors of these pathways need to be investigated. Particularly, the identity of the down-stream effectors of P13 kinase signaling and how PKC-s activates MEKs are not clear. The long-term career goal of the applicant is to investigate signaling pathways that control the proliferation and differentiation of hematopoietic cells. Therefore, the applicant proposes to study the role of P13 kinase in c-myc regulation in vivo and to identify the PI3 kinase pathway activated down stream molecules involved in the initiation of c-myc. The applicant will also identify the up-stream components of MEK-ERK and will study the role of Ras-Raf-1 signaling on the activation of c-myc and c-fos genes in erythroid cells. The methods involved in this proposal include genetic, molecular biological and biochemical approaches, such as analysis of transgenic mice, establishment of inducible stable cells, DNase I footprinting, EMSA, co-immunoprecipitation, kinase assays, Western-blot, RT-PCR, in vitro translation-GST-pull down and protein-DNA interaction affinity purification. The results of this research will help us to understand how cytokine-induced signaling control hematopoietic cell proliferation and differentiation.